The present invention is directed to a method of ordering plant embryos so that a specified end is always oriented in a given direction. The invention is also concerned with a method of separating viable from nonviable conifer somatic embryos and properly orienting the viable embryos for insertion into artificial seeds.
Reliable methods have now been developed for large scale production of plant somatic embryos of a large number of species using the techniques of tissue culture. In recent years much effort has been directed to development of techniques for embryogenesis of important conifer species. U.S. Pat. Nos. 4,957,866, 5,034,326, and 5,036,007 are exemplary of such methods. The work described in these patents has concentrated effort in particular on the commercially important United States species, loblolly pine Pinus taeda L. and Douglas-fir Pseudotsuga menziesii (Mirb.) Franco, and the similarly important European species, Norway spruce Picea abies (L.) Karst.
In culture the embryos are developed to a stage similar to the natural zygotic embryos occurring in mature seeds. For conifers these are very small, those for the species noted above falling in a range of about 2-4 mm in length. Embryos have a bipolar form which anticipates the ultimate plant. One end has a latent radicle or root and the other a whorl of tiny structures that will become the cotyledons of the germinated seed. The cotyledons, or seed leaves, are the first chlorophyll bearing organs and, in a natural seed, take over from the endosperm the job of providing nutrients to the newly germinated plant. The latent cotyledons look somewhat like a tiny crown situated at one end of the embryos.
Since somatic embryos lack the endosperm of the natural seed, some other means must be found to provide nutrients to the embryo at the time of germination. Various methods are available to accomplish this. The embryos may be placed on a solid germination medium containing the necessary carbohydrate and other nutrients, as described in any of the above patents. They may also be placed on a growing medium, or synthetic soil, which is saturated with an appropriate nutrient solution. In both of these cases sterility must be maintained until after the resulting plantlet is well established.
A preferred method of germinating a somatic embryo is to incorporate it into an artificial seed containing the essential nutrients. In essence, the artificial seed replaces the seed coat and endosperm of the natural seed. This method has the advantage that the embryos can be outplanted into a nursery bed in essentially the same manner as a natural seed.
A number of versions of artificial seeds have been described in the patent literature of the past half decade. Examples are U.S. Pat. Nos. 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent 1,241,552. Most of these are variations on the theme of completely encapsulating a somatic embryo within a hydrophilic gel. The gel may be based on a material such as sodium alginate which can then be insolubilized on only the surface or throughout by cation exchange with a material such as calcium chloride. By insolubilizing at least the surface the resulting artificial seed can be more readily handled.
In two of the above noted artificial seed patents, U.S. Pat. Nos. 4,615,141 and 4,777,762, the embryos are partially or completely dehydrated in the process. By completely dehydrated is meant desiccated to a moisture content in equilibrium with normal ambient room conditions.
Despite the significant amount of work that has already been done, none of the above proposed artificial seed structures has proved suitable for conifer embryos. Germination has been low when the embryo is completely enclosed within the gel droplet. A major advance in artificial seed construction is reported in U.S. patent application Ser. No. 604,656, filed Oct. 26, 1990, and commonly assigned with the present invention. In this improvement, the gel serving as the endosperm contains an oxygen carrier and is heavily oxygenated at the time of formation. Artificial seeds made by the procedures described therein, using conifer embryos, have shown high percentages of normal germination when placed on soil.
One problem to be dealt with in making any of the artificial seeds described above is insertion of the somatic embryos. In most of the issued patents the embryos are merely suspended in the gel former while in a fluid state. This is dispensed in a dropwise fashion with each drop presumably containing a single embryo. This fond hope has not been found to be the case in reality. Many of the droplets contained no embryos while others contained multiple embryos.
Another problem is the variation in embryo maturity typical in tissue cultures of somatic embryos. Some embryos are at a fully mature stage while others have not developed as well. The less well developed embryos will seldom germinate into a normal plantlet. The usual solution to this problem has been a tedious hand selection. More recently a technical paper was presented that describes separation of loblolly pine zygotic embryos and celery somatic embryos by maturity using sucrose solutions having varying density gradients (Velho, Saranga and Janick. Density separation of zygotic and somatic embryos. Abstract of paper given at the 87th Annual Meeting of the American Society of Horticultural Science, Tucson, Ariz., Nov. 4-8, 1990.) In the case of loblolly pine embryos, density declined during development to a relatively constant level of 1.031 g/mL on the 49th day after fertilization. As a corollary of this observation, density was seen to be inversely proportional to embryo length. In the case of celery embryos, those classified as mature and overmature showed the best conversion to normal seedlings after desiccation for 48 hours at 90% R.H. The Abstract was unclear as to whether desiccation followed density gradient separation or was carried out on hand selected embryos not subject to this treatment.
Published International Application WO 91/00781 describes a procedure and apparatus for separating cells such as plant embryos from culture medium. This employs a scanner to identify and determine location of the desired cell and a pipetting mechanism to remove them.
The preferred form of artificial seed described in the above noted patent application is far more sophisticated in construction than those using simple gel droplets. It requires insertion of the embryo, cotyledon end first, into a thin moisture pervious tube surrounded by a nutrient gel enclosed within an outer capsule which provides the mechanical equivalent of a seed coat. The tube into which the embryo is inserted is barely larger in diameter than the embryo itself. If an economical production rate is to be obtained this process must be mechanized as much as possible. The present invention is directed to a method of delivering viable embryos, oriented cotyledon end first, to a seed assembly point. No additional sorting or orientation is required. From the delivery location the embryos may be handled by robotic or other means for insertion into the inner tube of an artificial seed of the type noted above.